Role of tuberculosinyl metabolites in M. tuberculosis virulence Abstract Among all types of mycobacteria, Mycobacterium tuberculosis is the world's most prevalent and deadly pathogen. To find the particular molecules that enable its pathogenicity, we compared all detectable lipids in M. tuberculosis to those in a non-pathogenic vaccine strain (BCG). This subtractive screen identified a previously type of molecule called tuberculosinyl adenosine (TbAd) as well as the genes (Rv3377c, Rv3378c) that produce it. Although TbAd was overlooked in a century of tuberculosis research, our preliminary data show that TbAd is one of the most abundant lipids and is produced as six structurally related subfamilies. Published data show that M. tuberculosis is unique among mycobacteria in its ability to survive within the phagosomes of infected macrophages, based in its blockade of acid-mediated bacterial killing by macrophages. Our preliminary data show that TbAd is sufficient to block acidification of macrophage phagosomes. Therefore, we posit that TbAd and related tuberculosinyl metabolites are the long sought molecules that influence M. tuberculosis' unique ability to escape intracellular death. Here we propose to discover new tuberculosinyl metabolites in M. tuberculosis and determine their natural structures. Using synthetic TbAd and human macrophages, we will determine the cellular mechanism by which TbAd selectively inhibits phagosome acidification, while still allowing M. tuberculosis to be taken up into its phagosomal niche. Using M. tuberculosis lacking the biosynthetic enzymes (Rv 3378c, Rv3377c), we will measure the influence of TbAd in the outcome of natural infections. Last, we will test tuberculosinyl metabolites as targets for new diagnostic tests for human tuberculosis. Several features suggest that TbAd and TbAd-specific antibodies could be a highly specific chemical marker or infection. TbAd is abundantly secreted by M. tuberculosis, but is lacking in other pathogens that mimic tuberculosis. Therefore, we will detect TbAd and TbAd specific antibodies in serum and urine from a well-characterized patient cohort in Lima, Peru and in mice.